JP6412147B2 - Recombinant Escherichia microorganism having L-threonine producing ability and method for producing L-threonine using the same - Google Patents

Description

  The present invention relates to a recombinant microorganism belonging to the genus Escherichia which has been modified to express permease derived from coryneform bacteria and has an improved ability to produce L-threonine, and a method for producing L-threonine using the same.

  L-threonine is a kind of essential amino acid, widely used as a feed and food additive, and also used as an infusion for pharmaceuticals and a synthetic raw material for pharmaceuticals.

  L-threonine is produced mainly by fermentation using Escherichia, Sethcia, Prodencia, or Corynebacterium, which has been developed by an artificial mutation method or a gene recombination method, or an artificial mutant thereof. While genes related to threonine biosynthesis and various methods to increase their expression have been developed, there remains a need for methods that can produce L-threonine more economically and at higher yields.

  The GalP protein encoded by the galP gene in E. coli is known to be a galactose permease that transports various sugars including galactose and sucrose into the cell (V. Hernandez-Montalvo F. Valle F). Bolivar G. Gosset, Appl Microbiol Biotechnol (2001) 57: 186-191), which is also known to act as a glucose permease (Venter, Henrietta et al., Biochemical Journal 3: 2002). 243-252). There has been a report that increasing the expression of the galP gene in Escherichia coli through an increase in copy number or the like increases the threonine production ability (WO 2004/087937).

  In Corynebacterium glutamicum, it has been reported that inositol permease encoded by the iolT1 and iolT2 genes also acts as a glucose permease (Ikeda et al., Appl Microbiol Biotechnol (2011) 90: 1443). -1451), it has also been reported that these genes have high homology with the galP gene of Escherichia coli, but no report showing a relationship between the inositol permease and threonine production has yet been reported.

  The present inventors have found that when the iolT1 gene and / or iolT2 gene coding for inositol permease in Escherichia coli is incorporated into Escherichia coli, the Escherichia microorganism has an improved ability to produce L-threonine. The invention has been completed.

V. Hernandez-Montalvo F.R. Vale F.M. Bolivar G. Gosset, Appl Microbiol Biotechnol (2001) 57: 186-191 Venter, Henrietta et al. , Biochemical Journal (2002) 363: 243-252. Ikeda et al. , Appl Microbiol Biotechnol (2011) 90: 1443-1451

  Accordingly, an object of the present invention is to provide a recombinant Escherichia microorganism that has been modified to express a permease derived from coryneform bacteria and has an improved L-threonine-producing ability.

  Another object of the present invention is to provide a method for producing L-threonine at a high yield using the recombinant microorganism.

  In order to achieve the above object, the present invention provides a recombinant Escherichia microorganism transformed so that permease derived from Corynebacterium glutamicum is expressed.

  The present invention also provides a method for producing L-threonine at a high yield using the recombinant microorganism.

  According to the present invention, the growth rate of strains can be significantly improved compared to existing strains, and L-threonine can be produced at a high yield. Thereby, L-threonine productivity which has a big industrial meaning improves significantly.

FIG. 1 shows the homology alignment of the amino acid sequences of galP derived from E. coli and iolT1 and iolT2 derived from Corynebacterium glutamicum. FIG. 2 shows a cleavage map of the recombinant vector pCC1BAC-iolT1 containing the iolT1 gene derived from Corynebacterium glutamicum. FIG. 3 shows a cleavage map of the recombinant vector pCC1BAC-iolT2 containing the iolT2 gene derived from Corynebacterium glutamicum. FIG. 4 shows a cleavage map of the recombinant vector pCC1BAC-iolT1-iolT2 containing iolT1 and iolT2 genes derived from Corynebacterium glutamicum.

  The present invention is characterized by providing a recombinant microorganism belonging to the genus Escherichia which is transformed to express a permease derived from a coryneform bacterium and has an improved ability to produce L-threonine.

  In the present invention, the permease derived from the coryneform bacterium may be derived from Corynebacterium glutamicum, and may preferably be derived from Corynebacterium glutamicum ATCC13032. It is not limited.

  Most preferably, the permease derived from the coryneform bacterium may have the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. The permease derived from the coryneform bacterium having the amino acid sequence of SEQ ID NO: 1 is encoded by the iolT1 gene having the base sequence of SEQ ID NO: 3, and the permease derived from the coryneform bacterium having the amino acid sequence of SEQ ID NO: 2 is Encoded with the ioI T2 gene having the base sequence set forth in SEQ ID NO: 4.

  Moreover, as long as it is a protein having permease activity presented in the present invention, it is not less than 80%, preferably 90%, of a mutant having a partial mutation on the amino acid sequence of the protein or the amino acid sequence of the protein Permeases having a homology of at least%, more preferably at least 95%, particularly preferably at least 97% are also included in the present invention.

  In the present application, the term “homology” indicates identity between two amino acid sequences, and a person skilled in the art uses BLAST 2.0 to calculate parameters (parameters) such as score, identity, and similarity. Can be determined by methods well known to those skilled in the art.

  In one embodiment of the present invention, a gene having homology with the galP gene coding for glucose permease in E. coli is searched from Corynebacterium glutamicum ATCC13032, and as a result, the amino acid sequence encoded by galP of E. coli and the corynebacteria The amino acid sequence encoded by iolT1 (NCBI Reference Sequence: NC_006958.1, cg0223) derived from umglutamicum ATCC13032 shows 34% homology, and iolT2 (NCBI Reference Sequence: NC_006958.1, cg3387 is encoded by amino acid sequence). Shows 31% homology (see FIG. 1).

  The galP gene of E. coli is publicly known, and can also be obtained from the genome sequence of E. coli published by Blatner et al. (Science 277: 1453-1462 (1997)) (Accession no. AAC75876), and the National Center for Biotechnology Information NCBI It can also be obtained from databases such as the Japan DNA Data Bank (DDBJ).

  The Escherichia microorganism having the ability to produce L-threonine of the present invention may be Escherichia coli and an Escherichia coli mutant for producing L-threonine. More preferably, the Escherichia microorganism having the ability to produce L-threonine has methionine requirement, resistance to threonine analog, resistance to lysine analog, resistance to isoleucine analog and resistance to methionine analog, Escherichia coli KFCC 10718 (Korean Patent Registration No. 10-), which is an L-threonine-producing strain characterized in that two or more copies of a pyruvate carboxylase gene (phosphogene carboxylase gene, ppc gene) and a threonine operon are incorporated into the chromosome. E. coli KCCM 10541 (Korean Patent Registration No. 10-0576342) derived from No. 0058286).

  The method for expressing the gene encoding permease in the Escherichia microorganism having the ability to produce L-threonine of the present invention is not particularly limited. For example, a recombinant vector containing a gene encoding permease may be transformed into a microorganism, the copy number of the gene encoding permease may be increased, or the expression regulatory sequence of the gene encoding permease may be regulated. Of course, two or more methods may be used in combination. In general, as a method for increasing the expression level of a gene related to threonine biosynthesis, there is a method for increasing the number of genes possessed by one microorganism. For this purpose, a plasmid whose copy number is usually kept high is used. (Sambrook et al, Molecular cloning, 2nd edition, 1989, 1.3-1.5). In other words, by inserting a desired gene into a plasmid whose copy number is kept high, and transforming such a recombinant plasmid into a microorganism again, a gene copy corresponding to the number of copies of the plasmid per microorganism is obtained. The effect of increasing the number can be expected. A method of inserting a gene related to threonine biosynthesis into chromosomal DNA may be used.

  In one embodiment of the present invention, the present invention relates to a recombinant vector containing iolT1 gene and / or iolT2 gene derived from Corynebacterium glutamicum, and a recombinant having improved L-threonine production ability transformed into the recombinant vector. Provide Escherichia microorganisms.

  In the present application, the term “vector” refers to a nucleotide sequence of a polynucleotide that encodes the target protein operably linked to a suitable regulatory sequence so that the target protein is expressed in a suitable host. This refers to the DNA product contained. The regulatory sequences include a promoter capable of initiating transcription, any operator sequence to regulate such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that regulate the termination of transcription and decoding. After the vector has been transformed into a suitable host, it may replicate or function independently of the host genome or may be incorporated into the genome itself.

  The vector used in the present invention is not particularly limited as long as it can replicate in the host, and any vector known in the art can be used. Examples of commonly used vectors include native or recombinant plasmids, cosmids, viruses and bacteriophages.

  The present invention provides a recombinant vector containing the iolT1 gene derived from Corynebacterium glutamicum. Preferably, the recombinant vector is pCC1BAC-iolT1, and more preferably has the cleavage map of FIG.

  The present invention also provides a recombinant vector containing the iolT2 gene derived from Corynebacterium glutamicum. Preferably, the recombinant vector is pCC1BAC-iolT2, and more preferably has the cleavage map of FIG.

  Furthermore, the present invention provides a recombinant vector for simultaneously expressing the iolT1 and iolT2 genes derived from Corynebacterium glutamicum. Preferably, the recombinant vector is pCC1BAC-iolT1-iolT2, and more preferably has the cleavage map of FIG.

  In the present application, the term “transformation” means that a vector containing a polynucleotide encoding the target protein is taken into the host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. Say. As long as the transformed polynucleotide can be expressed in the host cell, it may be inserted into the host cell chromosome or located extrachromosomally. The polynucleotide may be incorporated in any form as long as it is incorporated into the host cell and can be expressed. The polynucleotide also includes DNA and RNA that encode the target protein. The polynucleotide may be incorporated in any form as long as it can be incorporated and expressed in the host cell. For example, the polynucleotide may be incorporated into a host cell in the form of an expression cassette, which is a polynucleotide construct that includes all the elements necessary to be expressed by itself. The expression cassette usually comprises a promoter operably linked to the gene, a transcription termination signal, a ribosome binding site and a translation termination signal. The expression cassette may be in the form of an expression vector that can replicate itself. The polynucleotide may be incorporated into the host cell in its own form and operably linked to a sequence required for expression in the host cell.

  In one aspect, the present invention provides a recombinant Escherichia microorganism having an improved ability to produce L-threonine transformed into a recombinant vector containing the iolT1 gene or iolT2 gene derived from Corynebacterium glutamicum.

  In another aspect, the present invention provides a recombinant Escherichia microorganism having improved L-threonine producing ability, transformed into a recombinant vector comprising the iolT1 gene and iolT2 gene derived from Corynebacterium glutamicum.

  Preferably, the transformed recombinant Escherichia microorganism of the present invention may be E. coli, preferably the microorganism is E. coli CA03-0230 (KCCM11370P), E. coli CA03-0260 (KCCM11369P) or E. coli CA03- 0231 (KCCM11371P) may be used.

  The above-described recombinant threonine producing strain of the present invention significantly improves the sugar depletion rate and growth rate of the strain by incorporating the iolT1 and / or iolT2 genes derived from coryneform bacteria into E. coli and increasing the permease expression in E. coli. By doing so, L-threonine can be produced at a high concentration.

  The present invention also provides a method for producing L-threonine, comprising the steps of culturing the transformed recombinant Escherichia microorganism and separating L-threonine from the culture solution of the microorganism.

  The recombinant Escherichia microorganism of the present invention is cultured by a usual method. Specifically, the microorganism can be inoculated and cultured in a culture medium containing a part or all of raw sugar or sucrose as a carbon source. The culturing process is performed using an appropriate medium and culturing conditions well known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Examples of the culture method include, but are not limited to, batch culture, continuous culture, and fed-batch culture. The medium used for culturing must appropriately meet the requirements of a particular strain.

  Specifically, the medium used in the present invention uses raw sugar or sucrose as the main carbon source, molasses containing a large amount of raw sugar can also be used as the carbon source, and other appropriate amounts of carbon source vary. Preferably, purified sucrose is used. Examples of nitrogen sources that can be used include organic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor and soybean wheat and inorganics such as iodine, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate A nitrogen source may be mentioned, and preferably peptone is used. These nitrogen sources can be used alone or in combination. The medium may contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salt as a phosphorus source. In addition, metal salts, such as magnesium sulfate or iron sulfate, may be included. In addition to these, amino acids, vitamins and suitable precursors may be included. These media or precursors can be added to the culture either batchwise or continuously.

  Specifically, the culture temperature is usually 27 ° C. to 37 ° C., preferably 30 ° C. to 37 ° C. The culture period continues to express the desired useful substance, and is preferably 10 to 100 hours.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

[ Comparative Example: Production of recombinant strain transformed into recombinant vector containing E. coli-derived galP gene and comparison of L-threonine productivity ]
In order to confirm the report (WO 2004/087937) that galactose permease plays the role of glucose permease in Escherichia coli and that the expression is increased by increasing the copy number (WO 2004/087937), L-threonine production was confirmed. Recombinant strains with an increased copy number of the galP gene were produced in Escherichia coli KCCM10541, and L-threonine productivity was evaluated.

(1) Production of recombinant vector containing E. coli-derived galP gene In order to obtain a 1.4 kb fragment containing an open reading frame of E. coli-derived galP gene, E. coli was used using a genomic tip system manufactured by Qiagen. The genomic DNA of the wild type strain W3110 was extracted.

  A chain polymerization reaction (hereinafter abbreviated as “PCR”) was performed using the gDNA as a template. The primers used at this time were SEQ ID NOs: 9 and 10, and PCR reaction conditions were as follows: denaturation at 94 ° C. for 30 seconds, annealing at 56 ° C. for 30 seconds, and extension at 72 ° C. 50 seconds, and this was repeated 30 times. The PCR product (hereinafter referred to as “galP fragment”) was obtained by eluting a band of a desired size after electrophoresis on a 0.8% agarose gel.

  After treating the obtained galP fragment with the restriction enzyme HindIII, the direction of the linear pCC1BAC vector (manufactured by EPICENTRE, hereinafter the same) treated with HindIII, which is the same restriction enzyme, coincides with the lac promoter on the vector. I was tied to.

  After transforming Escherichia coli DH5a cells to the prepared vector, it was smeared on a LB solid medium containing chloramphenicol and cultured at 37 ° C. overnight. After inoculating 3 ml of the LB liquid medium containing chloramphenicol with the cultured colony platinum ear, the plasmid DNA was recovered using a plasmid miniprep kit (manufactured by QIAGEN, hereinafter the same). . The size of the recombinant vector was confirmed by treatment with restriction enzyme HindIII (data not shown), denatured with primers of SEQ ID NOS: 11 and 12 for 30 seconds at 94 ° C., annealed at 56 ° C. for 30 seconds, and extended at 72 ° C. for 60 seconds. The clones were confirmed by PCR under the reaction conditions for 2 seconds. The recombinant vector was named pCC1BAC-galP (data not shown).

  In addition, the galP fragment was ligated so that the direction of the linear pCL1920 vector treated with HindIII, the same restriction enzyme, coincided with the lac promoter on the vector. After transforming Escherichia coli DH5a cells to the prepared vector, it was smeared on spectinomycin-containing LB solid medium and cultured at 37 ° C. overnight. The cultured colony-platinum ears were inoculated into 3 ml of spectinomycin-containing LB liquid medium and cultured overnight, and then plasmid DNA was recovered using a plasmid miniprep kit. The size of the recombinant vector was confirmed by treatment with the restriction enzyme HindIII (data not shown), denatured with primers of SEQ ID NOs: 13 and 14 for 30 seconds at 94 ° C., annealed at 56 ° C. for 30 seconds, and extended at 72 ° C. for 60 seconds. The clones were confirmed by PCR under the reaction conditions for 2 seconds. The recombinant vector was named pCL1920-galP (data not shown).

(2) Production of recombinant strain transformed into the recombinant vector The recombinant vector pCC1BAC-galP was incorporated into Escherichia coli KCCM10541 which is an L-threonine producing strain as a mother strain by electroporation, and chloramphenicol was 15 μg / ml. A single colony was selected by smearing on a solid medium containing. In the same manner as this method, the recombinant vector pCL1920-galP was taken into Escherichia coli KCCM10541 which is an L-threonine producing strain by electroporation, and smeared on a solid medium containing 50 μg / ml of spectinomycin. Colonies were selected.

  The selected strains were named KCCM10541 / pCC1BAC-galP and KCCM10541 / pCL1920-galP, respectively.

(3) Comparison of L-threonine productivity of recombinant strains The recombinant strain produced in (2) above was cultured in an Erlenmeyer flask using the threonine titer medium shown in Table 1 below in the same manner as described later, and L- Threonine productivity was confirmed.

  The titers were evaluated using Escherichia coli KCCM10541, KCCM10541 / pCC1BAC-galP and KCCM10541 / pCL1920-galP strains which are mother strains. When incorporated into each strain, the recombinant vector pCC1BAC-galP was expressed as 1 copy, and pCL1920-galP was expressed as 5 copies, and an attempt was made to confirm the effect accompanying an increase in the copy number.

The recombinant strains having different genetic traits are cultured overnight in an LB solid medium using a 33 ° C. incubator, and then are added to a 25 ml titer medium containing sucrose as shown in Table 1 above. After inoculating platinum ears one by one, they were cultured for 48 hours using an incubator at 33 ° C. and 200 rpm, and the results are shown in Table 2 below.

  As a result, as shown in Table 2, the mother strain E. coli KCCM10541 produced 32.0 g / L of L-threonine when cultured for 48 hours. L. threonine production was reduced to 31.1 g / L for certain E. coli KCCM10541 / pCC1BAC-galP, 30.8 g / L for KCCM10541 / pCL1920-galP, 0.9 g / L and 1.2 g / L, respectively, compared to the mother strain . As shown in Table 2, the E. coli KCCM10541 strain, the mother strain, showed a glucose consumption rate of 0.753 g / L / hr, while KCCM10541 / pCC1BAC-galP was 0.793 g / L / hr and KCCM10541 / pCL1920-galP. Indicates 0.816 g / L / hr, which is a sugar depletion rate improved by 5.3% and 8.4%, respectively, compared to the mother strain.

[ Example 1: Production of recombinant vector containing iolT1 and iolT2 genes derived from coryneform bacteria ]
(1) Comparison of homology with E. coli glucose permease galP The iolT1 and iolT2 genes encoding inositol permease in Corynebacterium glutamicum have almost the same homology as the galP gene encoding E. coli glucose permease. Have been reported. A gene having homology with E. coli-derived galP gene was found from the genome of wild-type Corynebacterium glutamicum ATCC 13032 and compared, and the results are shown in FIG.

  The amino acid sequence encoded by galP derived from E. coli and the amino acid sequence encoded by iolT1 derived from Corynebacterium glutamicum showed 34% homology, and the amino acid sequence encoded by iolT2 showed 31% homology.

(2) Preparation of iolT1 Gene Fragment In order to obtain a 1.5 kb fragment containing the open reading frame of the iolT1 gene of SEQ ID NO: 3, Corynebacterium glutamicum ATCC 13032 using a genomic-tip system manufactured by Qiagen. ) Genomic DNA was extracted.

  PCR was performed using the gDNA as a template. The primers used at this time were SEQ ID NOS: 5 and 6. PCR reaction conditions were denaturation at 94 ° C. for 30 seconds, annealing at 56 ° C. for 30 seconds, and extension at 72 ° C. for 60 seconds. Repeated times. The PCR product (hereinafter referred to as “iolT1 fragment”) was obtained by eluting a band of a desired size after electrophoresis on a 0.8% agarose gel.

(3) Production of recombinant vector pCC1BAC-iolT1 After the iolT1 fragment prepared in (2) above was treated with restriction enzyme HindIII, the linear pCC1BAC vector treated with HindIII, the same restriction enzyme, and the lac promoter on the vector were oriented Tied to match.

  Escherichia coli DH5a cells were transformed with the produced recombinant vector, and this was smeared on LB solid medium containing chloramphenicol and cultured at 37 ° C. overnight. The cultured colony platinum ear was inoculated into 3 ml of LB liquid medium containing chloramphenicol and cultured overnight, and then plasmid DNA was recovered using a plasmid miniprep kit. The size of the recombinant vector was confirmed by treatment with restriction enzyme HindIII (data not shown), denatured with primers of SEQ ID NOs: 11 and 12 for 30 seconds at 94 ° C., annealed at 56 ° C. for 30 seconds, and extended at 72 ° C. for 90 seconds. The clones were confirmed by PCR under the reaction conditions for 2 seconds. The recombinant vector was named pCC1BAC-iolT1.

(4) Preparation of iolT2 Gene Fragment In order to obtain a 1.6 kb fragment containing the open reading frame of the iolT2 gene of SEQ ID NO: 4, Corynebacterium glutamicum ATCC 13032 was used using a genomic-tip system manufactured by Qiagen. ) Genomic DNA was extracted.

  PCR was performed using the gDNA as a template. The primers used at this time were SEQ ID NOs: 7 and 8. PCR reaction conditions were denaturation at 94 ° C. for 30 seconds, annealing at 56 ° C. for 30 seconds, and extension at 72 ° C. for 60 seconds. Repeated times. The PCR product (hereinafter referred to as “iolT1 fragment”) was obtained by eluting a band of a desired size after electrophoresis on a 0.8% agarose gel.

(5) Production of recombinant vector pCC1BAC-iolT2 After treating the iolT2 fragment prepared in (4) with restriction enzyme EcoRI, the direction of the linear pCC1BAC vector treated with EcoRI, the same restriction enzyme, coincides with the lac promoter on the vector. I was tied to do.

  Escherichia coli DH5a cells were transformed with the produced recombinant vector, and this was smeared on LB solid medium containing chloramphenicol and cultured at 37 ° C. overnight. The cultured colony platinum ear was inoculated into 3 ml of LB liquid medium containing chloramphenicol and cultured overnight, and then plasmid DNA was recovered using a plasmid miniprep kit. The size of the recombinant vector was confirmed by treatment with restriction enzyme EcoRI (data not shown), denatured with primers of SEQ ID NOs: 11 and 12 for 30 seconds at 94 ° C., annealed at 56 ° C. for 30 seconds, and extended at 72 ° C. for 90 seconds. The clones were confirmed by PCR under the reaction conditions for 2 seconds. The recombinant vector was named pCC1BAC-iolT2.

(6) Production of recombinant vector pCC1BAC-iolT1-iolT2 After treating pCC1BAC-iolT2 vector produced in (5) above with restriction enzyme HindIII, olT1 fragment prepared in (2) above and lac promoter on the vector and direction Are tied to match.

  The prepared vector was transformed into E. coli DH5a cells, which were smeared on a LB solid medium containing chloramphenicol and cultured at 37 ° C. overnight. The cultured colony platinum ear was inoculated into 3 ml of LB liquid medium containing chloramphenicol and cultured overnight, and then plasmid DNA was recovered using a plasmid miniprep kit. The size of the recombinant vector was confirmed by treatment with restriction enzyme HindIII (data not shown), denatured with primers of SEQ ID NOS: 11 and 12 for 30 seconds at 94 ° C., annealed at 56 ° C. for 30 seconds, and extended at 72 ° C. for 120 seconds. The clones were confirmed by PCR under the reaction conditions for 2 seconds. The recombinant vector was named pCC1BAC-iolT1-iolT2.

[ Example 2: Production of transformed recombinant strain and comparison of L-threonine productivity ]
(1) Production of recombinant strain using wild-type Escherichia coli Recombinant vectors pCC1BAC-iolT1, pCC1BAC-iolT2 and pCC1BAC-iolT1-iolT2 produced in Example 1 were threonine operon and expression vector pBRThrABCR3 (Lee KH et al., Respectively). Incorporated into wild-type E. coli MG1655 containing Molecular Systems Biology (2007) 3: 149) by electroporation and smeared onto a solid medium containing 100 μg / ml ampicillin and 15 μg / ml chloramphenicol. Colonies were selected.

  The selected strains were named MG1655 / pBRThrABCR3 / pCC1BAC-iolT1, MG1655 / pBRThrABCR3 / pCC1BAC-iolT2 and MG1655 / pBRThrABCR3 / pCC1BAC-iolT1-iolT2, respectively.

These strains were confirmed for L-threonine productivity in the same manner as in Comparative Example 13 using a threonine titer medium having the composition shown in Table 1, and the results are shown in Table 3 below.

  As a result, as shown in Table 3, the E. coli MG1655 strain, the mother strain, produced 3.86 g / L of L-threonine when cultured for 48 hours, and the recombinant strain MG1655 / obtained in this Example was obtained. pCC1BAC-iolT1 produced 3.88 g / L, MG1655 / pCC1BAC-iolT2 and MG1655 / pCC1BAC-iolT1-iolT2 produced 3.92 g / L and 3.85 g / L L-threonine, respectively. That is, L-threonine at the same level as the mother strain was produced.

  As shown in Table 3, the wild type mother strain E. coli MG1655 strain showed a sugar depletion rate of 0.877 g / L / hr, while MG1655 / pCC1BAC-iolT2 was 1.035 g / L / hr, MG1655 / pCC1BAC-iolT1 showed 1.109 g / L / hr, and MG1655 / pCC1BAC-iolT1-iolT2 showed 1.123 g / L / hr. This is a sugar depletion rate improved by 18.0%, 26.5% and 28.1%, respectively, compared to the mother strain.

(2) Production of recombinant strain using Escherichia coli KCCM10541 Recombinant vectors pCC1BAC-iolT1, pCC1BAC-iolT2 and pCC1BAC-iolT1-iolT2 produced in Example 1 above were used as mother strains for E. coli KCCM10541, an L-threonine-producing strain. A single colony was selected by using a perforation method and smearing on a solid medium containing 15 μg / ml of chloramphenicol.

  The selected strains were named KCCM10541 / pCC1BAC-iolT1, KCCM10541 / pCC1BAC-iolT2, and KCCM10541 / pCC1BAC-iolT1-iolT2, respectively.

These strains were confirmed for L-threonine productivity in the same manner as in Comparative Example 13 using the threonine titer medium having the composition shown in Table 1 together with the recombinant microorganism obtained in Comparative Example 12. Is shown in Table 4 below.

  As a result, as shown in Table 4, the E. coli KCCM10541 strain, the mother strain, produced 30.3 g / L of L-threonine when cultured for 48 hours, and was obtained in Example 2-2 of the present invention. The recombinant strain KCCM10541 / pCC1BAC-iolT2 showed L-threonine productivity improved by 2.2 g / L over the mother strain, and KCCM10541 / pCC1BAC-iolT1 and KCCM10541 / pCC1BAC-iolT1-iolT2 were 29.5 g / L, respectively. 29.9 g / L of L-threonine was produced. That is, L-threonine at the same level as the mother strain was produced.

  Strains KCCM10541 / pCC1BAC-galP and KCCM10541 / pCL1920-galP with enhanced expression of the galP gene from E. coli produced 29.8 g / L and 29.3 g / L of L-threonine, respectively.

  As shown in Table 4, the E. coli KCCM10541 strain, the mother strain, showed a sugar depletion rate of 0.823 g / L / hr. -IolT2 shows 1.200 g / L / hr, KCCM10541 / pCC1BAC-iolT1 shows 1.213 g / L / hr, which is 32.8%, 45.8% and 47.4%, respectively, compared with the mother strain Improved sugar consumption rate. In addition, this indicates that when the sugar depletion rates of KCCM10541 / pCC1BAC-galP and KCCM10541 / pCL1920-galP were increased by 4.7% and 7.4%, respectively, compared to the mother strain, Corynebacterium ioI T1, iolT2 It can be confirmed that the sugar depletion rate of the gene-incorporated strain was significantly improved.

  The transformed Escherichia coli KCCM10541 / pCC1BAC-iolT1 was named CA03-0230, KCCM10541 / pCC1BAC-iolT2 was named CA03-0260, and KCCM10541 / pCC1BAC-iolT1-iolT2 was named CA03-0231. Deposited with microorganism storage sensors (Korean Culture Center of Microorganisms, hereinafter abbreviated as “KCCM”) under the accession numbers KCCM11370P, KCCM11369P and KCCM11371P, respectively.

  The above results show that E. coli-derived glucose permease expression is enhanced in E. coli having L-threonine-producing ability, that is, the permease expression from coryneform microorganisms is enhanced, that is, one or more iolT1 and iolT2 genes are incorporated. In this case, the rate of sugar consumption is increased, and it is proved that the time taken to produce the same concentration of threonine is shortened, that is, threonine productivity is increased.

  From the above description, those skilled in the art to which the present invention pertains understand that the present invention can be implemented in other specific forms without changing the technical idea and essential features. I can do it. In this connection, it should be understood that the above-described embodiments are illustrative in all aspects and not limiting. The scope of the present invention should be construed that the scope of the present invention includes any changes or modifications derived from the meaning and scope of the following claims and equivalents thereof rather than the above detailed description. is there.

[Trust number]
Depositary name: Korea Microbial Preservation Center (Overseas)
Accession number: KCCM11370P
Contract date: February 05, 2013

Depositary name: Korea Microbial Preservation Center (Overseas)
Accession number: KCCM11369P
Contract date: February 05, 2013

Depositary name: Korea Microbial Preservation Center (Overseas)
Accession number: KCCM11371P
Contract date: February 05, 2013

Claims (5)

A recombinant Escherichia microorganism having an improved L-threonine production rate, which has been transformed to contain the permease described in SEQ ID NO: 1 or SEQ ID NO: 2 derived from a coryneform bacterium. The recombinant Escherichia microorganism having an improved L-threonine production rate according to claim 1, wherein the recombinant Escherichia microorganism is Escherichia coli. SEQ ID NO: 1 and derived from coryneform of SEQ ID NO: 2 permease is transformed to include also both the set seen recombinant Escherichia microorganism according to claim 1. The recombinant Escherichia microorganism is Escherichia coli, the pair saw recombinant Escherichia microorganism of claim 3. Inoculating and culturing the microorganism according to any one of claims 1 to 3,
Separating L-amino acids from the culture;
A method for producing L-threonine, comprising:

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